Inositolphosphoglycan and ribose for treatment of ischaemia-reperfusion injury

ABSTRACT

Compositions comprising inositolphosphoglycans (IPGs) and ribose are disclosed, and their use in the prevention or treatment of ischaemic-reperfusion injury. This treatment increases the energy generating systems of cells by employing the mitochondrial oxidative restoration system. The use of the compositions in preserving organs for transplantation is also disclosed.

FIELD OF THE INVENTION

The present invention relates to material and methods relating to theprevention or treatment of ischaemia-reperfusion injury, and inparticular to compositions comprising inositolphosphoglycans (IPGS) andtheir medical use in the prevention or treatment of ischaemia.

BACKGROUND OF THE INVENTION

The search for novel therapies for ischaemic-reperfusion injury in theheart has been a subject of intense research, both for recovery fromopen-heart surgery, where the limited capacity for the heart to surviveischaemia is a well researched problem (Stanley et al, 1997), and fromthe viewpoint of modulating the extent of damage incurred duringepisodes of cardiac ischaemia (Stanley et al, 1997). It is also wellestablished that the incidence of coronary heart disease is a majorfactor in the morbidity and mortality of diabetic patients (Fuller etal, 1983; Hillier et al, 1988). There is also evidence that standarddrugs for the treatment of diabetes of the sulphonylurea group may havenegative effects, including those on K⁺ channel function (smits & Thien,1995; Muhlhauser et al, 1997).

The complexity of the events following ischaemia-reperfusion is suchthat there is a very wide ranging database of potential therapeutic andcardioplegic agents targeting differing aspects of the cascade leadingto damage to cardiac function. It has been apparent from work as earlyas the 1960s (Danforth et al, 1960; Berne, 1963) to the present (Zimmer,1996; Houston et al, 1997) that a key feature of the cascade ofinterlinked biochemical events following ischaemic-reperfusion injurycentres on the loss of adenine nucleotides from the myocardium. Thereis, thus, an absolute requirement for the restitution of theintracellular ATP concentration and the energy charge of the cell inorder to restore normal cardiac function.

Adenine nucleotide synthesis can occur via utilization or reutilisationof adenine nucleotide breakdown products via the salvage pathway, or viade novo synthesis from small molecular weight precursors. The former isthe most effective in terms of energy requirement (Mangano, 1997;Meldrum et al, 1997).

However, in addition to the requirement for the purine ring, a supply ofphosphoribosylpyrophosphate (PRPP) is essential both for the salvage andde novo routes of synthesis; this latter compound is, in turn, subjectto tight regulation and is dependent upon a supply of ribose-5-phosphate(Kunjara et al, 1987). Zimmer (1980) demonstrated that restitution ofmyocardial adenine nucleotides was accelerated by ribose, as was thenormalisation of depressed heart function in rats (Zimmer, 1983). Thisauthor stated that “The advantage of ribose over other metabolicinterventions is that is does not affect the haemodynamics of the heartwith an ultimate change in oxygen demand and that is has no vasoactiveproperties which may result in afterload alterations”.

Recently, Zimmer (1996) reported that in two in vivo rat models, theoverloaded and catecholamine-stimulated heart and the infarcted heart,the normalisation of the cardiac adenine nucleotide pool by ribose wasaccompanied by improvement in global heart function. Further, thecombined treatment with ribose and adenine or inosine inisoproteronal-treated rats was more effective in the restoration andcompletely restored the ATP level within a shorter period of time thaneither treatment alone.

SUMMARY OF THE INVENTION

While the results showing the effect of repletion of cardiac ATP areencouraging, the prior art approaches described above suffer from thedisadvantage that the biosynthetic pathways themselves require ATP, asdoes the reconversion of AMP to ADP and ATP, the required ATP being thevery compound in short supply. Further, as mentioned above, thecomplexity of the biochemistry associated with ischaemia means that itis not clear from the prior art how alternative approaches could avoidthis problem.

The present invention relates to the finding that inositolphosphoglycans(IPGs), and in particular P-type IPGs, or their synthetic analogues, canbe used to generate ATP from ADP while helping to avoid the productionof toxic byproducts and helping to minimise the ATP requirement for theprocess. Thus, compositions comprising IPGs can be used to prevent ortreat ischaemia-reperfusion, in particular in conditions where there isa reduction or risk of reduction in cellular ATP levels, e.g. in cardiacischaemia, in surgery (especially heart or transplant surgery), inpreserving organs for transplantation, in the treatment of stroke and asan anti-apoptosis agent to protect against cell death (especially inmuscle cells).

Accordingly, in a first aspect, the present invention provides acomposition for treating an ischaemic-reperfusion injury, thecomposition comprising an inositolphosphoglycan (IPG) or an IPGsynthetic analogue, and ribose.

In a further aspect, the present invention provides the use of aninositolphosphoglycan (IPG) for the preparation of a medicament for thetreatment of ischaemic-reperfusion injury.

The IPGs present in the medicament can be P- or A-type IPGs, orsynthetic analogues of them. The production of IPGs and IPG analogues isdiscussed further below. Preferably, the IPG is a P-type IPG or a P-typesynthetic analogue.

The present invention is based on the realisation that an alternativeapproach to the problem of increasing the energy generating systems ofthe cell is to employ the mitochondrial oxidative restoration system, inparticular by the regulation of the key enzyme for the entry of pyruvateinto the tricarboxylic acid cycle, pyruvate dehydrogenase. Accordingly,the present proposal centres upon the use of naturally occurringactivators of pyruvate dehydrogenase phosphatase, theinositolphosphoglycans, to promote the conversion of pyruvatedehydrogenase to the active form, thereby enhancing therephosphorylation of AMP and ADP.

Advantageously, the composition includes one or more other components,in combination with the IPGs, for use in the treatment ofischaemia-reperfusion injury as described herein. Among the agents to beused in combination with IPGs from different sources are:

-   (1) Adenosine and purine compounds as precursors of ATP and as    modulators of TNFα action (see Bouchard & Lamontagne, 1998; de Jong    et al, 1997; Meldrum et al, 1997).-   (2) Ribose as a precursor of PRPP (see Kunjara et al, 1987; Zimmer,    1996).-   (3) Nicotinamide and derivatives to prevent the loss of NAD and ATP    by inhibition of poly-ADP ribose synthetase (see Bromme & Holz,    1996; Zingarelli et al, 1996; Gilad et al, 1997; Thiememann et al,    1997).-   (4) ca²⁺uptake inhibitors (see Ferrari et al, 1996; Loh et al, 1998;    Russ et al, 1996).-   (5) Addition of IPGs to established cardioplegic solutions (see    Choong and Gavin, 1996; Bozkurt et al, 1997).-   (6) Maintenance of glutathione systems (see Konorev et al, 1996).    Glutathione in its reduced form (GSH) is an important factor in the    prevention of damage by hydrogen peroxide. Hydrogen peroxide is a    component of ischaemia-reperfusion injury and protection is afforded    by the action of glutathione peroxidase and GSH. The importance of    GSH and the pentose phosphate pathway in the chain reactions    protecting the cell from free radical damage is illustrated in FIG.    1 from Zubairu et al, 1983.-   (7) Endothelin inhibitors (see Goodwin et al, 1997; Pernow & Wang,    1997). Endothelin-1 (ET-1) is an extremely potent vasoconstrictor    peptide derived from vascular endothelial cells. During and    following myocardial ischaemia and reperfusion, the myocardial    production and release of ET-1 is stimulated and the coronary    constriction to ET-1 is enhanced. The pathophysiological role for    ET-1 in the development of ischaemia has a strong basis and the    potential for cardioprotective effects of ET-1 antagonists has been    considered by Pernow and Wang (1997).

Ischaemia-reperfusion injury can arise in a wide range of conditions andthe medicament can be used to treat these conditions. Examples includeischaemia resulting from myocardial infarct, during surgery (especiallyopen heart surgery, or during organ transplantation, e.g. employing themedicament as a cardioplegia solution for heart or lung bypass surgery),and in stroke. The medicament can also be used to ameliorate the effectsof ischaemia in tissues, in particular as an anti-apoptotic agent toprevent cell death following ischaemia, e.g. muscle cell death.

In a further aspect, the present invention provides a method forpreserving an organ for transplantation, the method comprising exposingthe organ with a composition comprising an inositolphosphoglycan (IPG),and optionally one or more of the components mentioned above. Asischaemia is common in organs for transplantation, this approach isuseful for preserving the energy level present in the organ prior totransplantation and during surgery. Conveniently, the composition can beperfused through the organ or used to store the organ prior totransplantation, i.e. be a storage medium for the organ.

In a further aspect, the present invention provides compositionscomprising a P-type IPG and ribose. In these compositions, the IPGdrives mitochondrial oxidation and results in ATP generation from ADPwithout production of toxic byproducts. Preferably, the compositionadditionally comprises a purine or purine nucleotide precursor toprovide the basic structural element of ATP. Other possible componentsof the composition are described above.

This composition is useful in organ preservation, in general surgery(e.g. as a perfusion fluid) and in other situations for the preventionor treatment of ischaemia in cells. Preferably, the composition issupplied as a powder or concentrate from which a liquid composition canbe prepared. Alternatively, the composition can be supplied ready to usein as a liquid. Formulations and optional ingredients of the compositionare discussed further below.

In further aspects, the present invention provides above compositionsfor use in a method of medical treatment, for example in the preparationof a medicament for the treatment of ischaemic conditions discussedabove.

Embodiments of the present invention will now be described by way ofexample and not by limitation with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the correlation between the hepatic PRPP concentration andthe log of ribose 5-phosphate and the flux through the oxidative pentosephosphate assay pathway (C1-C6) in different dietary and hormonalconditions in rats.

FIG. 2 shows the correlation between the hepatic PRPP concentration andATP and energy charge (EC), free cytosolic NAD⁺/NADH and NAD⁺/NADPH indifferent dietary and hormonal conditions in rats.

FIG. 3 shows the correlation between the hepatic PRPP concentration andADP, AMP and Pi in different dietary and hormonal conditions in rats.

FIGS. 4A and 4B shows the steady state concentration and the effect ofinsulin on extractable IPG A-type from the heart and other tissues fromadult male rats. FIG. 4A shows the results of a lipogenesis assay andFIG. 4B shows a CAMP-dependent protein kinase A assay. The solid columnsshow results in the absence of insulin, while the hatched columns showresults 2 minutes after injection with insulin. 1 unit is the amount ofA-type IPG causing a 50% increase in the basal rate of lipogenesis or a50% decrease in the activity of cAMP dependent protein kinase.

FIGS. 4C and 4D show the steady state concentration and the effect ofinsulin on extractable IPG P-type from heart and other tissues fromadult male rats. FIG. 4C shows a PDH phosphatase assay and FIG. 4D showsa cAMP-dependent protein kinase-P assay. The solid columns show resultsin the absence of insulin, while the hatched columns show results 2minutes after injection with insulin. 1 unit is the amount of P-type IPGcausing a 50% increase in the activity of PDH phosphatase or a 50%decrease in the activity of cAMP dependent protein kinase.

FIGS. 4E and 4F show the results of a thymidine incorporation into EGFreceptor transfected 3T3 cells, plotted against IPG A-type and IPGP-type concentrations respectively.

FIG. 5 shows a schematic setting out the role of ribose, IPGs andselected substrates on the prevention or recovery from ischaemic damageaccording to the present invention.

FIG. 6 shows a schematic setting out the site of action of IPG P-type inthe activation of the PDH complex.

DETAILED DESCRIPTION OF THE INVENTION

IPGs and IPG Analogues

Studies have shown that A-type mediators modulate the activity of anumber of insulin-dependent enzymes such as cAMP dependent proteinkinase (inhibits), adenylate cyclase (inhibits) and cAMPphospho-diesterases (stimulates). In contrast, P-type mediators modulatethe activity of insulin-dependent enzymes such as pyruvate dehydrogenasephosphatase (stimulates), glycogen synthase phosphatase (stimulates) andcAMP dependent kinase (inhibits). The A-type mediators mimic thelipogenic activity of insulin on adipocytes, whereas the P-typemediators mimic the glycogenic activity of insulin on muscle. Both A-andP-type mediators are mitogenic when added to fibroblasts in serum freemedia. The ability of the mediators to stimulate fibroblastproliferation is enhanced if the cells are transfected with theEGF-receptor. A-type mediators can stimulate cell proliferation in thechick cochleovestibular ganglia.

Soluble IPG fractions having A-type and P-type activity have beenobtained from a variety of animal tissues including rat tissues (liver,kidney, muscle brain, adipose, heart) and bovine liver. A- and P-typeIPG biological activity has also been detected in human liver andplacenta, malaria parasitized RBC and mycobacteria. The ability of ananti-inositolglycan antibody to inhibit insulin action on humanplacental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPGaction on rat diaphragm and chick cochleovestibular ganglia suggestscross-species conservation of many structural-features. However, it isimportant to note that although the prior art includes these reports ofA- and P-type IPG activity in some biological fractions, thepurification or characterisation of the agents responsible for theactivity is not disclosed.

A-type substances are cyclitol-containing carbohydrates, also containingZn²⁺ion and optionally phosphate and having the properties of regulatinglipogenic activity and inhibiting CAMP dependent protein kinase. Theymay also inhibit adenylate cyclase, be mitogenic when added toEGF-transfected fibroblasts in serum free medium, and stimulatelipogenesis in adipocytes.

P-type substances are cyclitol-containing carbohydrates, also containingM²⁺ and/or Zn²⁺ ions and optionally phosphate and having the propertiesof regulating glycogen metabolism and activating pyruvate dehydrogenasephosphatase. They may also stimulate the activity of glycogen synthasephosphatase, be mitogenic when added to fibroblasts in serum freemedium, and stimulate pyruvate dehydrogenase phosphatase.

Methods for obtaining A-type and P-type IPGs are set out in Caro et al,1997 and in WO98/11116 or WO98/11117. The present invention can employIPGs found in nature, for instance in tissues such a liver or placentafrom animals such as human, pig, rat or other animals), and obtainedusing methods described in the above applications. These IPGs arepreferably purified from the tissues, and more preferably purified tohomogeneity. As defined herein, “substantially purified” describes IPGswhich have been separated from components which are naturally presentwith the IPGs in the source tissue. Preferably, the compositions are atleast 75%, more preferably at least 90%, more preferably at least 95%,and still more preferably at least 99% by weight of IPGs.

Alternatively or additionally, the present invention can employcyclitol-containing IPG analogues, e.g. inositol-containing IPGanalogues. These compounds have the advantage that they can be morereadily prepared using synthetic organic chemistry methods, rather thanbeing extracted from natural source materials. Preferred P-typesynthetic analogues contain chiro-inositol, or a derivative thereof, asa structural unit or motif, and have one or more of the properties ofP-type IPGs indicated above, especially activation of pyruvatedehydrogenase phosphatase. An example of a chiro-inositol containing IPGanalogue is compound C4,1D-6-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-chiro-inositol 1-phosphatewhich can be synthesised as described in Jaramillo et al, 1994.

Preferred A-type synthetic analogues contain myo-inositol, or aderivative thereof, as a structural unit or motif and have one or moreof the properties of A-type IPGs indicated above. An example of amyo-inositol containing IPG analogue is compound C31D-6-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1,2-(cyclicphosphate), which can be been prepared as described in Zapata et al,1994.

Pharmaceutical Compositions

The compositions of the invention can be formulated according to thespecific application which the composition is intended to treat. Thecompositions may comprise, in addition to the one or more IPGs, andoptionally one or more of the above components, a pharmaceuticallyacceptable excipient, carrier, buffer, stabiliser or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient(s). The precise nature of the carrier or other material maydepend on the route of administration, e.g. intravenous, cutaneous orsubcutaneous, nasal, intramuscular, intraperitoneal routes. Forembodiments in which the medicaments or compositions of the inventionare used in organ preservation, they can be formulated so that they aresuitable for storing or perfusing organs or tissue.

The compositions may be supplied in the form of a powder or concentratefrom which a composition can be prepared. Alternatively, the compositionmay be supplied in a ready to use form, e.g. as a liquid. In eitherevent, the composition may include other active ingredients, adjuvantsor carriers. Thus, physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol may be included.

In embodiments in which the composition is used in the prophylactic ortherapeutic treatment of conditions associated with a risk of ischaemia,preferably the composition is administered to a patient via intravenous,cutaneous or subcutaneous injection, or injection at the site ofaffliction. In this case, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as sodium chloride injection, Ringer's injection,lactated Ringer's injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.Injection is a preferred mode of delivery for compositions for treatingischaemia that results from myocardial infarction, stroke or to treat orprotect against apoptosis.

The active ingredients in the composition are preferable administered toan individual in preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Experimental

Experiments in this laboratory have shown with rat heart preparationsthat the tissue PRPP concentration in anoxic conditions fell and waspartially restored by addition of ribose to the medium. Perhaps ofgreater significance was our observation of the decline in cellular PRPPin a range of tissues, including heart, in experimental diabetes (seeTable 1). These data suggest that ribose or a ribose precursor and/orpurine derivatives could advantageously be included in the medicamentscompositions of the invention.

While reported effects of repletion of cardiac ATP are encouraging, itis apparent that these biosynthetic processes themselves require ATP, asdoes the reconversion of AMP to ADP and ATP, the required ATP being thevery compound in short supply. Thus, any mechanism increasing the energygenerating systems of the cell, primarily and most effectively via themitochondrial oxidative restoration, would be advantageous to theprocess of cellular restoration. In this context, the regulation of thekey enzyme for the entry of pyruvate into the tricarboxylic acid cycle,the pyruvate dehydrogenase complex, must be considered.

This enzyme is highly regulated by, among other factors, the energystatus of the cell, by the NADH/NAD+ ratio and by the acetyl COA/CoAratio, via the interconversion of active/inactive forms of pyruvatedehydrogenase by phosphorylation/dephosphorylation reactions regulatedby pyruvate dehydrogenase kinase and regulation of this enzyme complexat the pyruvate crossroads. This system operates in a manner such thatischaemic conditions activate PDH kinase dehydrogenase and so shut offenergy production at this step. In order to circumvent this inhibition,even in ischaemia, it is necessary to activate the PDH phosphatase andthis can be accomplished by the presence of IPGs. Pyruvate dehydrogenaseactivity is the most important determinant of whether pyruvate isconverted to lactate, leading to lactic acidosis and a low level of ATPfrom glycolysis, or whether the highly efficient ATP generating systemof the tricarboxylic acid cycle will be facilitated.

The present invention centres upon the use of naturally occurringactivators of pyruvate dehydrogenase phosphatase, theinositolphosphoglycans, to promote the conversion of pyruvatedehydrogenase to the active form (Rademacher et al, 1994; Varela-Nietoet al, 1998), thereby enhancing the rephosphorylation of AMP and ADP.The preferred combination of purine nucleotide precursors (to providethe basic structural element of the required ATP), together with ribose(to provide the ribose 5-phosphate for PRPP formation) andinositolphosphoglycans (to shift the pyruvate dehydrogenase complextowards the active form, generate energy and decrease lactic acidosis)can be used to treat ischaemic conditions, e.g. ischaemic heartconditions, and the loss of ATP. As can be seen from FIG. 5, such atherapy would supply all three major elements required for therestoration of the energy charge of the cell.

-   (1) Ribose, as the precursor of the synthesis of the adenine lost    from the cell during extended ischaemia;-   (2) PRPP, an essential component of the adenine biosynthetic    pathway; and,-   (3) An increase energy yield from carbohydrate fuel which can    provide the energy needed for biosynthetic processes in (1) and (2)    and also to rephosphorylate such ADP and AMP as remains in the cell    to ATP.

Therefore, the approach of using inositolphosphoglycans either alone ortogether with other precursors of adenine nucleotide synthesis andcompounds protecting against loss of ATP (e.g. by inhibition of poly ADPribose), in the treatment of ischaemic conditions in heart, kidney,brain or other organs, is a fundamental new approach to attempting tolimit cell damage. In a preferred embodiment of the invention, thecombination of ribose, purine precursors and nicotinamide, the latter toprevent lost of NAD and ATP by inhibition of polyADP ribose synthase,with the inositolphosphoglycans, the potent second messenger systemfunctioning in the regulation of proteinphosphorylation/dephosphorylation cycles, is a multifaceted attack onthe very basis of cellular damage in ischaemic conditions, that is theloss of ATP.

Table 1 demonstrates that in diabetes, there is a drop in tissue levelsof PRPP. This drop could make diabetic patients more at risk ofmorbidity following an ischaemic attack. It is well established thatboth the incidence and complications of coronary heart disease areelevated in diabetic patients and decreased tissue levels of PRPP couldbe the crucial link. Thus, the present invention is particularly suitedto the treatment of ischaemic conditions arising from diabetes. FIG. 1demonstrates that tissue levels of ribose 5-phosphate are important inmaintaining PRPP levels and FIG. 5 shows that ribose is the directprecursor of ribose 5-phosphate. Therefore, one important component inmaintaining high levels of PRPP is to provide ribose as the precursorfor ribose 5-phosphate.

FIGS. 2 and 3 demonstrate that in order to have high levels of PRPP intissues, the cellular energy charge must be high. Under anoxicconditions, this is difficult since the enzyme PDH kinase is activated.The action of this enzyme is to inactivate the PDH complex, which isinvolved in the biosynthesis of acetyl-CoA and NADH. The NADH sogenerated in the reperfusion period is oxidized by the electrontransport chain to generate ATP. The acetyl-CoA is a substrate for theKrebs cycle in which one glucose can be oxidized to 36 ATPs via thegeneration of further NADH. The action of IPG-P type mediators is toactivate PDH phosphatase which counteracts the PDH kinase and allows foractivation of the PDH complex. This activation is shown in FIG. 6. Theaction of the IPG-P type and the amounts recovered from various tissuesbefore and after insulin infusion are shown in FIGS. 4C and D. Inparticular, an increase in activity is found in muscle and kidney uponinsulin infusion. In contrast, decreased activity is found in heart,adipose tissue and brain (FIG. 4C). These data demonstrate that aninsulin infusion could not substitute for a direct infusion of the IPG-Ptype. FIG. 5 shows that an insulin infusion will also affect the IPG-Aactivity differentially in tissues and this effect would not occur oninfusion of just IPG-P compound or its analogues.

TABLE 1 EFFECTS OF EXPERIMENTAL DIABETES ON PHOSPHORIBOSYL PYROPHOSPHATE(PRPP) CONTENT OF HEART AND OTHER TISSUES PHOSPHORIBOSYL PYROPHOSPHATECONTENT (nmoles/g tissue) STZ Diabetic Tissue Control (14 Days) “P”Heart 3.61 ± 0.11 2.60 ± 0.20 <0.01 (15) (6) Liver 10.5 ± 0.64 7.60 ±0.43 <0.001 (17) (5) Lung 5.40 ± 0.05 3.44 ± 0.39 <0.001 (16) (5) Testis 5.0 ± 0.30 2.5 ± 0.9 <0.02 (20) (5) Blood glucose  7.0 ± 0.45  28 ± 3.0<0.001 (mM) (25) (7) Body weight 309 ± 17  226 ± 21  <0.01 (g) (20) (7)

The tissues were freeze-clamped and the PRPP content estimated asdescribed by Kunjara et al (1987). The values are given as means±SEM;Fisher's P values are given. The adult male rats were used 14 days afterthe induction of diabetes with streptozotocin.

REFERENCES

The following references are cited to show the state of the art.

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1. A composition comprising an inositolphosphoglycan (IPG) or an IPGcontaining chiro-inositol or myo-inositol and ribose, wherein the IPGcontaining chiro-inositol or myo-inositol has the ability to activatepyruvate dehydrogenase phosphatase.
 2. The composition of claim 1wherein the IPG is a P-type IPG.
 3. The composition of claim 1 whereinIPG containing chiro-inositol is a P-type IPG.
 4. The composition ofclaim 1, further comprising adenosine or purine.
 5. The composition ofclaim 1 or 2, wherein the composition is a liquid composition.
 6. Thecomposition of claim 1 or 2, wherein the composition is a powder orconcentrate from which a liquid composition can be prepared.
 7. Thecomposition of claim 1 or 2, further comprising a pharmaceuticallyacceptable excipient.